PROJECT SUMMARY Membrane traffic, an essential cellular process that plays a role in many human diseases, requires key biophysical steps including formation of membrane buds, loading of these buds with specific molecular cargo, separation from the parent membrane, and fusion with the target membrane. The prevailing view has been that structured protein motifs drive these processes. However, many proteins that contain these structural motifs also contain large intrinsically disordered protein (IDP) domains of 300-1500 amino acids, including most clathrin adaptor proteins and COPII coat components. While these IDP domains have been regarded primarily as flexible binding and recruitment motifs, the principal investigator's laboratory has recently reported that IDPs are highly efficient drivers of membrane remodeling in their own right. Further, preliminary data for the proposed work demonstrate that when IDP domains bind membrane surfaces in sufficient numbers, they serve as strong drivers of membrane fission. How can molecules without a defined structure drive membrane bending and fission? Substantial preliminary data in this application supports the working hypothesis that disordered domains are highly effective drivers of membrane remodeling through the mechanism of protein crowding. Specifically, the work of the principal investigator, supported by findings from others, has recently revealed that collisions among membrane-bound proteins generate entropic pressure that provides a potent driving force for membrane deformation. IDPs are particularly efficient generators of entropic pressure owing to their large hydrodynamic radii and the substantial energetic cost of extending them. Building on preliminary findings, the goal of the proposed work is to elucidate the physical roles of IDPs in membrane traffic by measuring the impact of entropic pressure on key steps of the process. Work in Aim 1 will elucidate the physical mechanisms that IDPs use to generate entropic pressure, testing the working hypothesis that it depends on steric, electrostatic, and lipid-mediated interactions. Work in Aim 2 will measure the contribution of IDPs to membrane fission, testing the working hypothesis that IDP domains generate entropic pressure that dramatically reduces the energetic cost of membrane fission. Finally, work in Aim 3 will use disordered polymers to assess and control receptor selection by trafficking vesicles, testing the working hypothesis that entropic pressure among bulky receptors opposes endocytic uptake and can be used as a tool to drive accumulation of receptors at the plasma membrane. The significance of this work lies in its potential to change how we think about the molecules and mechanisms that control membrane traffic. Specifically, while current models focus on specific structural domains thought to sculpt membrane surfaces, this work suggests that proteins that lack defined structure, IDPs, may be among the most potent drivers of membrane traffic. Further, understanding how entropic pressure influences membrane traffic will create new opportunities to control the process, providing a set of physical tools for manipulating receptor recycling and signaling.